Epithelial sodium channels (ENaC) in the apical membrane of the collecting duct (CD) are co-localised with a variety of ATP-activated P2 receptors. Intraluminal ATP can inhibit ENaC-mediated Na⁺ reabsorption (Unwin et al. 2003), but there is controversy concerning the P2 receptor subtype responsible. In vitro studies in mouse CD implicate the P2Y₂ receptor, but in vivo studies in the rat suggest otherwise (Shirley et al. 2005). Here we have used immunohistochemistry to identify those P2 receptor subtypes present in the apical membrane of the rat CD, and patch-clamp studies to investigate pharmacologically which of these might be responsible for inhibition of ENaC-mediated Na⁺ reabsorption. Kidneys from terminally anaesthetised adult Sprague Dawley rats, that had been maintained on a low (0.01%) sodium diet (in order to increase CD ENaC expression), were perfusion-fixed with paraformaldehyde (4%). Using 8 µm kidney slices from 3 rats and antibodies specific for P2X₁-P2X₇ receptor subunits and P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁ and P2Y₁₂ receptors, as well as for α-, β- and γ-ENaC subunits, strong immunoflourescence indicated the co-localisation of P2X₁, P2X₂, P2X₄, P2X₆ and P2Y₄ receptors with ENaC in the CD apical membrane. In further experiments, microdissected CD segments, from rats treated similarly but not perfusion-fixed, were split open to expose the apical membrane of individual principal cells and studied under voltage-clamp conditions using the whole-cell perforated-patch technique. We tested a range of P2 agonists for their activity and for their effect on amiloride (10 μM)-sensitive (i.e. ENaC-mediated) currents. The nucleotides (10 µM) ATP, ATPγS, 2meSATP (all broad-spectrum P2 agonists), Ap₆A (P2X₁ selective) and UTP (P2Y₂ and P2Y₄ selective) all elicited inward currents (amplitude ~500 pA; V_h = -60 mV; n=3). Furthermore, with the exception of UTP, they reduced subsequent amiloride-sensitive current-voltage relationship amplitude (n=3) without changing the inward rectification or reversal potential. These data are consistent with ionotropic P2X receptor-mediated inhibition of ENaC in the rat CD. Candidate P2X subunits are P2X₁, P2X₂, P2X₄ and/or P2X₆.